Monoclonal Antibodies to Hepatocyte Growth Factor

ABSTRACT

The present invention is directed toward a neutralizing monoclonal antibody to hepatocyte growth factor, a pharmaceutical composition comprising same, and methods of treatment comprising administering such a pharmaceutical composition to a patient.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/825,060 filed Apr. 15, 2004, which claims the benefit of theprovisional application U.S. patent application Ser. No. 60/464,061filed Apr. 18, 2003, both of which are incorporated by reference intheir entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to the combination of monoclonalantibody (mAb) and recombinant DNA technologies for developing novelbiologics, and more particularly, for example, to the production ofmonoclonal antibodies that bind to and neutralize Hepatocyte GrowthFactor.

BACKGROUND OF THE INVENTION

Human Hepatocyte Growth Factor (HGF) is a multifunctional heterodimericpolypeptide produced by mesenchymal cells. HGF has been shown tostimulate angiogenesis, morphogenesis and motogenesis, as well as thegrowth and scattering of various cell types (Bussolino et al., J. Cell.Biol. 119:629, 1992; Zarnegar and Michalopoulos, J. Cell. Biol.129:1177, 1995; Matsumoto et al., Ciba. Found. Symp. 212:198, 1997;Birchmeier and Gherardi, Trends Cell. Biol. 8:404, 1998; Xin et al. Am.J. Pathol. 158:1111, 2001). The pleiotropic activities of HGF aremediated through its receptor, a transmembrane tyrosine kinase encodedby the proto-oncogene cMet. In addition to regulating a variety ofnormal cellular functions, HGF and its receptor c-Met have been shown tobe involved in the initiation, invasion and metastasis of tumors(Jeffers et al., J. Mol. Med. 74:505, 1996; Comoglio and Trusolino, J.Clin. Invest. 109:857, 2002). HGF/cMet are coexpressed, oftenover-expressed, on various human solid tumors including tumors derivedfrom lung, colon, rectum, stomach, kidney, ovary, skin, multiple myelomaand thyroid tissue (Prat et al., Int. J. Cancer 49:323, 1991; Chan etal., Oncogene 2:593, 1988; Weidner et al., Am. J. Respir. Cell. Mol.Biol. 8:229, 1993; Derksen et al., Blood 99:1405, 2002). HGF acts as anautocrine (Rong et al., Proc. Natl. Acad. Sci. USA 91:4731, 1994;Koochekpour et al., Cancer Res. 57:5391, 1997) and paracrine growthfactor (Weidner et al., Am. J. Respir. Cell. Mol. Biol. 8:229, 1993) andanti-apoptotic regulator (Gao et al., J. Biol. Chem. 276:47257, 2001)for these tumors.

HGF is a 102 kDa protein with sequence and structural similarity toplasminogen and other enzymes of blood coagulation (Nakamura et al.,Nature 342:440, 1989; Weidner et al., Am. J. Respir. Cell. Mol. Biol.8:229, 1993, each of which is incorporated herein by reference) (FIG.1). Human HGF is synthesized as a 728 amino acid precursor (preproHGF),which undergoes intracellular cleavage to an inactive, single chain form(proHGF) (Nakamura et al., Nature 342:440, 1989; Rosen et al., J. Cell.Biol. 127:1783, 1994). Upon extracellular secretion, proHGF is cleavedto yield the biologically active disulfide-linked heterodimeric moleculecomposed of an α-subunit and β-subunit (Nakamura et al., Nature 342:440,1989; Naldini et al., EMBO J. 11:4825, 1992). The a-subunit contains 440residues (69 kDa with glycosylation), consisting of the N-terminalhairpin domain and four kringle domains. The β-subunit contains 234residues (34 kDa) and has a serine protease-like domain, which lacksproteolytic activity. Cleavage of HGF is required for receptoractivation, but not for receptor binding (Hartmann et al., Proc. Natl.Acad. Sci. USA 89:11574, 1992; Lokker et al., J. Biol. Chem. 268:17145,1992). HGF contains 4 putative N-glycosylation sites, 1 in the α-subunitand 3 in the β-subunit. HGF has 2 unique cell specific binding sites: ahigh affinity (Kd=2×10⁻¹⁰ M) binding site for the cMet receptor and alow affinity (Kd=10⁻⁹ M) binding site for heparin sulfate proteoglycans(HSPG), which are present on the cell surface and extracellular matrix(Naldini et al., Oncogene 6:501, 1991; Bardelli et al., J. Biotechnol.37:109, 1994; Sakata et al., J. Biol. Chem., 272:9457, 1997). NK2 (aprotein encompassing the N-terminus and first two kringle domains of theα-subunit) is sufficient for binding to cMet and activation of thesignal cascade for motility, however the full length protein is requiredfor the mitogenic response (Weidner et al., Am. J. Respir. Cell. Mol.Biol. 8:229, 1993). HSPG binds to HGF by interacting with the N terminusof HGF (Aoyama, et al., Biochem. 36:10286, 1997; Sakata, et al., J.Biol. Chem. 272:9457, 1997). Postulated roles for the HSPG-HGFinteraction include the enhancement of HGF bioavailability, biologicalactivity and oligomerization (Bardelli, et al., J. Biotechnol.37:109,1994; Zioncheck et al., J. Biol. Chem. 270:16871, 1995).

cMet is a member of the class IV protein tyrosine kinase receptorfamily. The full length cMet gene was cloned and identified as the cMetproto-oncogene (Cooper et al., Nature 311:29, 1984; Park et al., Proc.Natl. Acad. Sci. USA 84:6379, 1987). The cMet receptor is initiallysynthesized as a single chain, partially glycosylated precursor,p170^((MET)) (FIG. 1) (Park et al., Proc. Natl. Acad. Sci. USA 84:6379,1987; Giordano et al., Nature 339:155, 1989; Giordano et al., Oncogene4:1383, 1989; Bardelli et al., J. Biotechnol. 37:109, 1994). Uponfurther glycosylation, the protein is proteolytically cleaved into aheterodimeric 190 kDa mature protein (1385 amino acids), consisting ofthe 50 kDa α-subunit (residues 1-307) and the 145 kDa β-subunit. Thecytoplasmic tyrosine kinase domain of the β-subunit is involved insignal transduction.

Several different approaches have been investigated to obtain anantagonistic molecule of the HGF/cMet interaction: truncated HGFproteins such as NK1 (N terminal domain plus kringle domain 1; Lokker etal., J. Biol. Chem. 268:17145, 1993), NK2 (N terminal domain pluskringle domains 1 and 2; Chan et al., Science 254:1382, 1991) and NK4(N-terminal domain plus four kringle domains; Kuba et al., Cancer Res.60:6737, 2000), anti-cMet mAbs (Dodge, Master's Thesis, San FranciscoState University, 1998) and anti-HGF mAbs (Cao et al., Proc. Natl. Acad.Sci. USA 98:7443, 2001, which is incorporated herein by reference).

NK1 and NK2 can compete effectively with the binding of HGF to itsreceptor, but have been shown to have partial agonistic activities invitro (Cioce et al., J. Biol. Chem. 271:13110, 1996; Schwall et al., J.Cell Biol. 133:709, 1996), rather than purely antagonist activities asdesired. More recently, Kuba et al., Cancer Res. 60:6737, 2000,demonstrated that NK4 could partially inhibit the primary growth (FIG.2) and metastasis of murine lung tumor LLC in a nude mouse model bycontinuous infusion of NK4. The fact that NK4 had to administeredcontinuously to obtain a partial growth inhibition of primary tumorsindicates a potentially short half-life of the NK4 molecule and/or lackof potency. Compared to NK4, the approach of using antibodies willbenefit from their favorable pharmacokinetics and the possibility ofobtaining antibodies with much higher potency.

As another approach, Dodge (Master's Thesis, San Francisco StateUniversity, 1998) generated antagonistic anti-cMet monoclonal antibodies(mAbs). One mAb, 5D5, exhibited strong antagonistic activity in ELISA,but induced a proliferative response of cMet-expressing BAF-3 cells,presumably due to dimerization of the membrane receptors. Prat et al.,J. Cell Sci. 111:237, 1998, also reported such agonistic activities ofanti-cMet mAbs. Zaccolo et al., Eur. J. Immunol 27:618, 1997, used phagedisplay methods do develop human Fab fragments against mouse and humanhepatocyte growth factor. These Fab fragments had no effect on theactivity of HGF when used alone. When one of the anti-human HGF Fabfragments was combined with an antibody that bound to the Fab fragmentitself, it actually enhanced the activity of HGF in a biological assay.

Cao et al., Proc. Natl. Acad. Sci. USA 98:7443, 2001, demonstrated thatthe administration of a cocktail of three anti-HGF mAbs, which wereselected based upon their ability to inhibit the scattering activity ofHGF in vitro, were able to inhibit the growth of human tumors in thexenograft nude mouse model (FIG. 3). They postulated that three mAbsrecognizing three different binding sites on HGF were required toinhibit the bioactivities of HGF in vivo: two mAbs inhibited the bindingof HGF to cMet and one mAb inhibited the binding of HGF to heparin.However, it is impractical for commercial and regulatory reasons todevelop a drug combining three novel mAbs, e.g., because some clinicalactivity of each antibody would need to be demonstrated independently.

Thus, there is a need for a single monoclonal antibody that blocksbiological activity of HGF in vitro and in vivo. The present inventionfulfills this and other needs.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the invention provides a neutralizing mAb to humanHepatocyte Growth Factor (HGF). The mAb inhibits at least one, andpreferably several or all biological activities of HGF including bindingto its receptor cMet, inducing scattering of cells such as Madin-Darbycanine kidney cells, inducing proliferation of 4MBr-5 monkey epithelialcells and/or hepatocytes and/or HUVEC, and inducing angiogenesis. TheAnti-HGF mAb can inhibit such an activity when used as a single agent. Apreferred anti-HGF mAb inhibits, most preferably completely inhibits,growth of a human tumor xenograft in a mouse. Preferably, the mAb of theinvention is chimeric, humanized, human-like or human. Exemplaryantibodies are L2G7 and its chimeric and humanized forms. Cell linesproducing such antibodies are also provided. In another embodiment, apharmaceutical composition comprising a neutralizing anti-HGF antibody,e.g., chimeric or humanized L2G7, is provided. In a third embodiment,the pharmaceutical composition is administered to a patient to treatcancer or other disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic models of HGF and cMet.

FIG. 2. Graph showing that NK4 partially inhibits the primary growth ofmurine lung tumor LLC in nude mice (from Kuba et al., Cancer Res.60:6737, 2000). NK4 was infused continuously for 14 days from 4^(th) dayafter tumor implantation s.c. in nude mice.

FIG. 3. Graph showing that a cocktail of three anti-HGF mAbs is requiredto inhibit the growth of human brain tumor U-118 cells in nude mice(from Cao et al., Proc. Natl. Acad. Sci. USA 98:7443, 2001). U-118 tumorcells were injected s.c. into nude mice. From day 1 anti-HGF mAbs A-1,-5, and -7, or mAbs 7-2 and -3 were administered at 200 μg/injection,twice/wk for 10 wks.

FIG. 4. Determination of relative binding epitopes of mAbs L1H4, L2C7,L2G7 using competitive binding ELISA. Plates were coated withrecombinant HGF (rHGF), blocked with skim milk and incubated withsuboptimal concentration of biotinylated mAbs in the presence of 100×excess amounts of unlabeled mAbs. Biotinylated mAb bound was detected bythe addition of HRP-Strepavidin.

FIG. 5. Binding of anti-HGF mAbs to rHGF as determined in a direct HGFbinding ELISA. Plate was coated with the HI-F11 supernatant containingrHGF, blocked by 2% skim milk and incubated with mAbs, followed by theaddition of HRP-GaMIgG (as described under Examples).

FIG. 6. Abilities of anti-HGF mAbs to capture rHGF-Flag in solution.Anti-HGF mAbs were captured on a goat anti-mouse IgG coated ELISA plate.Plates were then blocked with 2% skim milk and incubated with rHGF-Flag,followed by HRP-M2 anti-Flag mAb (as described under Examples).

FIG. 7. Inhibition of rHGF-Flag binding to cMet-Fc by anti-HGF mAbs in acapture ELISA. cMet-Fc captured on goat anti-human IgG coated plate isincubated with HGF-Flag preincubated with/without mAbs. The boundrHGF-Flag was detected by the addition of HRP-M2 anti-Flag mAb (asdescribed under Examples).

FIG. 8. Neutralization of HGF induced MDCK scattering by anti-HGF mAbL2G7. (A) Control without any treatment. (B) rHGF+IgG. (C) rHGF+mAbL2G7. MDCK cells were incubated with a 1:20 dilution of H1-F 11 culturesupernatant (˜3 μg/ml of HGF) in the presence of 10 μg/ml of mAbs.Photos were taken at 100× magnification.

FIG. 9. Inhibition of HGF-induced proliferation of Mv 1 LU cells by L2G7mAb. The fold molar excess of mAb over HGF is shown on the horizontalaxis, and the cpm×10⁻² incorporated is shown on the vertical axis. Datapoints were obtained in triplicate.

FIG. 10. Inhibition of HGF-induced proliferation of HUVEC by L2G7 mAband control mouse antibody (mIgG). Data points were obtained intriplicate.

FIG. 11. Effect on HGF-induced proliferation of HCT 116 colon tumorcells by L2G7 and L1H4 antibodies. Data point were obtained intriplicate.

FIG. 12. Effect of treatment with L2G7 mAb or PBS (control) on growth ofU-118 tumors in groups of NIH III Beige/Nude mice (n=6). Arrow indicateswhen injections began. (A) Tumor size vs day from tumor implantation.(B) Tumor mass at end of experiment.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides neutralizing anti-HGF monoclonal antibodies,pharmaceutical compositions comprising them, and methods of using themfor the treatment of disease.

1. Antibodies

Antibodies are very large, complex molecules (molecular weight of˜50,000 or about 1320 amino acids) with intricate internal structure. Anatural antibody molecule contains two identical pairs of polypeptidechains, each pair having one light chain and one heavy chain. Each lightchain and heavy chain in turn consists of two regions: a variable (“V”)region involved in binding the target antigen, and a constant (“C”)region that interacts with other components of the immune system. Thelight and heavy chain variable regions fold up together in 3-dimensionalspace to form a variable region that binds the antigen (for example, areceptor on the surface of a cell). Within each light or heavy chainvariable region, there are three short segments (averaging 10 aminoacids in length) called the complementarity determining regions(“CDRs”). The six CDRs in an antibody variable domain (three from thelight chain and three from the heavy chain) fold up together in 3-Dspace to form the actual antibody binding site which locks onto thetarget antigen. The position and length of the CDRs have been preciselydefined. Kabat, E. et al., Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services, 1983, 1987. Thepart of a variable region not contained in the CDRs is called theframework, which forms the environment for the CDRs.

A monoclonal antibody (mAB) is a single molecular species of antibodyand therefore does not encompass polyclonal antibodies produced byinjecting an animal (such as a rodent, rabbit or goat) with an antigen,and extracting serum from the animal. A humanized antibody is agenetically engineered (monoclonal) antibody in which the CDRs from amouse antibody (“donor antibody”, which can also be rat, hamster orother similar species) are grafted onto a human antibody (“acceptorantibody”). Humanized antibodies can also be made with less than thecomplete CDRs from a mouse antibody (e.g., Pascalis et al., J. Immunol.169:3076, 2002). Thus, a humanized antibody is an antibody having CDRsfrom a donor antibody and variable region framework and constant regionsfrom a human antibody. Thus, typically a humanized antibody comprises(i) a light chain comprising three CDRs from a mouse antibody, e.g.,L2G7, a variable region framework from a human antibody, and a humanconstant region, and (ii) a heavy chain comprising three CDRs from amouse antibody, e.g., L2G7, a variable region framework from a humanantibody and a human constant region. In addition, in order to retainhigh binding affinity, at least one of two additional structuralelements can be employed. See, U.S. Pat. No. 5,530,101 and U.S. Pat. No.5,585,089, each of which is incorporated herein by reference, whichprovide detailed instructions for construction of humanized antibodies.

In the first structural element, the framework of the heavy chainvariable region of the humanized antibody is chosen to have maximalsequence identity (between 65% and 95%) with the framework of the heavychain variable region of the donor antibody, by suitably selecting theacceptor antibody from among the many known human antibodies. Sequenceidentity is determined when antibody sequences being compared arealigned according to the Kabat numbering convention. In the secondstructural element, in constructing the humanized antibody, selectedamino acids in the framework of the human acceptor antibody (outside theCDRs) are replaced with corresponding amino acids from the donorantibody, in accordance with specified rules. Specifically, the aminoacids to be replaced in the framework are chosen on the basis of theirability to interact with the CDRs. For example, the replaced amino acidscan be adjacent to a CDR in the donor antibody sequence or within 4-6angstroms of a CDR in the humanized antibody as measured in3-dimensional space.

A chimeric antibody is an antibody in which the variable region of amouse (or other rodent) antibody is combined with the constant region ofa human antibody; their construction by means of genetic engineering iswell-known. Such antibodies retain the binding specificity of the mouseantibody, while being about two-thirds human. The proportion of nonhumansequence present in mouse, chimeric and humanized antibodies suggeststhat the immunogenicity of chimeric antibodies is intermediate betweenmouse and humanized antibodies. Other types of genetically engineeredantibodies that may have reduced immunogenicity relative to mouseantibodies include human antibodies made using phage display methods(Dower et al., WO91/17271; McCafferty et al., WO92/001047; Winter,WO92/20791; and Winter, FEBS Lett. 23:92, 1998, each of which isincorporated herein by reference) or using transgenic animals (Lonberget al., WO93/12227; Kucherlapati WO91/10741, each of which isincorporated herein by reference).

As used herein, the term “human-like” antibody refers to a Mab in whicha substantial portion of the amino acid sequence of one or both chains(e.g., about 50% or more) originates from human immunoglobulin genes.Hence, human-like antibodies encompass but are not limited to chimeric,humanized and human antibodies. As used herein, a“reduced-immunogenicity” antibody is one expected to have significantlyless immunogenicity than a mouse antibody when administered to humanpatients. Such antibodies encompass chimeric, humanized and humanantibodies as well as antibodies made by replacing specific amino acidsin mouse antibodies that may contibute to B- or T-cell epitopes, forexample exposed residues (Padlan, Mol. Immunol. 28:489, 1991). As usedherein, a “genetically engineered” antibody is one for which the geneshave been constructed or put in an unnatural environment (e.g., humangenes in a mouse or on a bacteriophage) with the help of recombinant DNAtechniques, and would therefore, e.g., not encompass a mouse mAb madewith conventional hybridoma technology.

The epitope of a mAb is the region of its antigen to which the mAbbinds. Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1×, 5×, 10×, 20× or 100× excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay compared to a control lackingthe competing antibody (see, e.g., Junghans et al., Cancer Res. 50:1495,1990, which is incorporated herein by reference). Alternatively, twoantibodies have the same epitope if essentially all amino acid mutationsin the antigen that reduce or eliminate binding of one antibody reduceor eliminate binding of the other. Two antibodies have overlappingepitopes if some amino acid mutations that reduce or eliminate bindingof one antibody reduce or eliminate binding of the other.

2. Neutralizing anti-HGF Antibodies

A monoclonal antibody (mAb) that binds HGF (i.e., an anti-HGF mAb) issaid to neutralize HGF, or be neutralizing, if the binding partially orcompletely inhibits one or more biological activities of HGF (i.e., whenthe mAb is used as a single agent). Among the biological properties ofHGF that a neutralizing antibody may inhibit are the ability of HGF tobind to its cMet receptor, to cause the scattering of certain cell linessuch as Madin-Darby canine kidney (MDCK) cells; to stimulateproliferation of (i.e., be mitogenic for) certain cells includinghepatocytes, 4MBr-5 monkey epithelial cells, and various human tumorcells; or to stimulate angiogenesis, for example as measured bystimulation of human vascular endothelial cell (HUVEC) proliferation ortube formation or by induction of blood vessels when applied to thechick embryo chorioallantoic membrane (CAM). Antibodies of the inventionpreferably bind to human HGF, i.e., to the protein encoded by theGenBank sequence with Accession number D90334 (incorporated byreference).

A neutralizing mAb of the invention at a concentration of, e.g., 0.01,0.1, 0.5, 1, 2, 5, 10, 20 or 50 μg/ml will inhibit a biological functionof HGF (e.g., stimulation of proliferation or scattering) by about atleast 50% but preferably 75%, more preferably by 90% or 95% or even 99%,and most preferably approximately 100% (essentially completely) asassayed by methods described under Examples or known in the art.Inhibition is considered complete if the level of activity is within themargin of error for a negative control lacking HGF. Typically, theextent of inhibition is measured when the amount of HGF used is justsufficient to fully stimulate the biological activity, or is 0.05, 0.1,0.5, 1, 3 or 10 μg/ml. Preferably, at least 50%, 75%, 90%, or 95% oressentially complete inhibition will be achieved when the molar ratio ofantibody to HGF is 0.5×, 1×, 2×, 3×, 5× or 10×. Preferably, the mAb willbe neutralizing, i.e., inhibit the biological activity, when used as asingle agent, but possible 2 mAbs will be needed together to giveinhibition. Most preferably, the mAb will neutralize not just one butseveral of the biological activities listed above; for purposes herein,an anti-HGF mAb that used as a single agent neutralizes all thebiological activities of HGF will be called “fully neutralizing”, andsuch mAbs are most preferable. MAbs of the invention will preferably bespecific for HGF, that is they will not bind, or only bind to a muchlesser extent (e.g., Ka at least ten-fold less), proteins that arerelated to HGF such as fibroblast growth factor (FGF) and vascularendothelial growth factor (VEGF). Preferred antibodies lack agonisticactivity toward HGF. That is, the antibodies block interaction of HGHwith cMet without stimulating cells bearing HGF directly. MAbs of theinvention typically have a binding affinity (K_(a)) for HGF of at least10⁷ M⁻¹ but preferably 10⁸ M⁻¹ or higher, and most preferably 10⁹ M⁻¹ orhigher or even 10¹⁰ M−1 or higher.

MAbs of the invention include anti-HGF antibodies in their naturaltetrameric form (2 light chains and 2 heavy chains) and may be of any ofthe known isotypes IgG, IgA, IgM, IgD and IgE and their subtypes, i.e.,human IgG1, IgG2, IgG3, IgG4 and mouse IgG1, IgG2a, IgG2b, and IgG3. ThemAbs of the invention are also meant to include fragments of antibodiessuch as Fv, Fab and F(ab′)₂; bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17:105, 1987), single-chainantibodies (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879, 1988;Bird et al., Science 242:423, 1988); and antibodies with alteredconstant regions (e.g., U.S. Pat. No.5,624,821). The mAbs may be ofanimal (e.g., mouse, rat, hamster or chicken) origin, or they may begenetically engineered. Rodent mAbs are made by standard methodswell-known in the art, comprising multiple immunization with HGF inappropriate adjuvant i.p., i.v., or into the footpad, followed byextraction of spleen or lymph node cells and fusion with a suitableimmortalized cell line, and then selection for hybridomas that produceantibody binding to HGF, e.g., see under Examples. Chimeric andhumanized mAbs, made by art-known methods mentioned supra, are preferredembodiments of the invention. Human antibodies made, e.g., by phagedisplay or transgenic mice methods are also preferred (see e.g., Doweret al., McCafferty et al., Winter, Lonberg et al., Kucherlapati, supra).More generally, human-like, reduced immunogenicity and geneticallyengineered antibodies as defined herein are all preferred.

The neutralizing anti-HGF mAbs L1H4, L2C7 and L2G7 mAbs described infraare examples of the invention, with L2G7 a preferred example.Neutralizing mAbs with the same or overlapping epitope as any of thesemAbs, e.g., as L2G7, provide other examples. A chimeric or humanizedform of L2G7 or with LGF is an especially preferred embodiment. A mAb(including chimeric, humanized and human antibodies) that competes withL2G7 for binding to HGF and neutralizes HGF in at least one, andpreferably all, in vitro or in vivo assays described herein is alsopreferred. MAbs that are 90%, 95%, 99% or 100% identical (determined byaligning antibody sequences according to the Kabat convention) to L2G7in amino acid sequence, at least in the CDRs are included in theinvention. Preferably such antibodies differ from L2G7 by a small numberof functionally inconsequential amino acid substitutions (e.g.,conservative substitutions), deletions, or insertions. Preferably suchantibodies retain the functional properties of L2G7, i.e., suchantibodies neutralize HGF in at least one, and preferably all, in vitroor in vivo assays described herein. For purposes of classifying aminoacids substitutions as conservative or nonconservative, amino acids maybe grouped as follows: Group I (hydrophobic sidechains): norleucine,met, ala, val, leu, ile; Group II (neutral hydrophilic side chains):cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basicside chains): asn, gln, his, lys, arg; Group V (residues influencingchain orientation): gly, pro; and Group VI (aromatic side chains): trp,tyr, phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

Native mAbs of the invention may be produced from their hybridomas.Genetically engineered mAbs, e.g., chimeric or humanized mAbs, may beexpressed by a variety of art-known methods. For example, genes encodingtheir light and heavy chain V regions may be synthesized fromoverlapping oligonucleotides and inserted together with available Cregions into expression vectors (e.g., commercially available fromInvitrogen) that provide the necessary regulatory regions, e.g.,promoters, enhancers, poly A sites, etc. Use of the CMVpromoter-enhancer is preferred. The expression vectors may then betransfected using various well-known methods such as lipofection orelectroporation into a variety of mammalian cell lines such as CHO ornon-producing myelomas including Sp2/0 and NS0, and cells expressing theantibodies selected by appropriate antibiotic selection. See, e.g., U.S.Pat. No. 5,530,101. Larger amounts of antibody may be produced bygrowing the cells in commercially available bioreactors.

Once expressed, the mAbs or other antibodies of the invention may bepurified according to standard procedures of the art such asmicrofiltration, ultrafiltration, protein A or G affinitychromatography, size exclusion chromatography, anion exchangechromatography, cation exchange chromatography and/or other forms ofaffinity chromatography based on organic dyes or the like. Substantiallypure antibodies of at least about 90 or 95% homogeneity are preferred,and 98% or 99% or more homogeneity most preferred, for pharmaceuticaluses.

3. Therapeutic Methods

In a preferred embodiment, the present invention provides apharmaceutical formulation comprising the antibodies described herein.That is, the antibodies can be used in the manufacture of a medicamentfor treatment of disease. Pharmaceutical formulations (i.e.,medicaments) of the antibodies contain the mAb in a physiologicallyacceptable carrier, optionally with excipients or stabilizers, in theform of lyophilized or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,or acetate at a pH typically of 5.0 to 8.0, most often 6.0 to 7.0; saltssuch as sodium chloride, potassium chloride, etc. to make isotonic;antioxidants, preservatives, low molecular weight polypeptides,proteins, hydrophilic polymers such as polysorbate 80, amino acids,carbohydrates, chelating agents, sugars, and other standard ingredientsknown to those skilled in the art (Remington's Pharmaceutical Science16^(th) edition, Osol, A. Ed. 1980). The mAb is typically present at aconcentration of 1-100 mg/ml, e.g., 10 mg/ml.

Antibodies of the invention are typically substantially pure fromundesired contaminant. This means that the antibody is typically atleast about 50% w/w (weight/weight) pure, as well as being substantiallyfree from interfering proteins and contaminants. Preferably theantibodies are at least 90, 95% or 99% w/w pure. Pharmaceuticalcompositions for parenteral administration are usually sterile,substantially isotonic and prepared in accordance with GoodManufacturing Practices of the FDA or similar body.

In another preferred embodiment, the invention provides a method oftreating a patient with a disease using an anti-HGF mAb in apharmaceutical formulation. The mAb prepared in a pharmaceuticalformulation can be administered to a patient by any suitable route,especially parentally by intravenous infusion or bolus injection,intramuscularly or subcutaneously. Intravenous infusion can be givenover as little as 15 minutes, but more often for 30 minutes, or over 1,2 or even 3 hours. The mAb can also be injected directly into the siteof disease (e.g., a tumor), or encapsulated into carrying agents such asliposomes. The dose given will be sufficient to alleviate the conditionbeing treated (“therapeutically effective dose”) and is likely to be 0.1to 5 mg/kg body weight, for example 1, 2, 3 or 4 mg/kg, but may be ashigh as 10 mg/kg or even 15 or 20 mg/kg. A fixed unit dose may also begiven, for example, 50, 100, 200, 500 or 1000 mg, or the dose may bebased on the patient's surface area, e.g., 100 mg/M². Usually between 1and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) are administered to treatcancer, but 10, 20 or more doses may be given. The mAb can beadministered daily, biweekly, weekly, every other week, monthly or atsome other interval, depending, e.g. on the half-life of the mAb, for 1week, 2 weeks, 4 weeks, 8 weeks, 3-6 months or longer. Repeated coursesof treatment are also possible, as is chronic administration. A regimeof a dosage and intervals of administration that alleviates or at leastpartially arrests the symptoms of the disease (biochemical, histologicand/or clinical), including its complications and intermediatepathological phenotypes in development of the disease is referred to asa therapeutically effective regime.

The pharmaceutical compositions of the invention can also be used inprophylaxis of a patient at risk of cancer. Such patients include thosehaving genetic susceptibility to cancer, patients who have undergoneexposure to carcinogenic agents, such as radiation or toxins, andpatients who have undergone previous treatment for cancer and are atrisk of recurrence. A prophylactic dosage is an amount sufficient toeliminate or reduce the risk, lessen the severity, or delay the outsetof the disease, including biochemical, histologic and/or clinicalsymptoms of the disease, its complications and intermediate pathologicalphenotypes presenting during development of the disease. Administrationof a pharmaceutical composition in an amount and at intervals effectiveto effect one or more of these objects is referred to as aprophylactically effective regime.

Diseases especially susceptible to therapy with the anti-HGF mAbs ofthis invention include solid tumors known or suspected to requireangiogenesis or to be associated with elevated levels of HGF, forexample ovarian cancer, breast cancer, lung cancer (small cell ornon-small cell), colon cancer, prostate cancer, pancreatic cancer, renalcancer, gastric cancer, liver cancer, head-and-neck tumors, melanoma,sarcomas, and brain tumors (e.g., glioblastomas), of children or adults.Treatment can also be administered to patients having leukemias orlymphomas. In a preferred embodiment, the anti-HGF mAb can beadministered together with (i.e., before, during or after) otheranti-cancer therapy. For example, the anti-HGF mAb may be administeredtogether with any one or more of the chemotherapeutic drugs known tothose of skill in the art of oncology, for example Taxol (paclitaxel) orits derivatives, platinum compounds such as carboplatin or cisplatin,anthrocyclines such as doxorubicin, alkylating agents such ascyclophosphamide, anti-metabolites such as 5-fluorouracil, or etoposide.The anti-HGF mAb can be administered in combination with two, three ormore of these agents in a standard chemotherapeutic regimen, for exampletaxol and carboplatin, e.g. for breast and ovarian cancer. Other agentswith which the anti-HGF mAb can be administered include biologics suchas monoclonal antibodies, including Herceptin™ against the HER2 antigen,Avastin™ against VEGF, or antibodies to the EGF receptor, as well assmall molecule anti-angiogenic or EGF receptor antagonist drugs. Inaddition, the anti-HGF mAb can be used together with radiation therapyor surgery.

Treatment (e.g., standard chemotherapy) including the anti-HGF mAbantibody may increase the median progression-free survival or overallsurvival time of patients with these tumors (e.g., ovarian, breast,lung, pancreas, brain and colon, especially when relapsed or refractory)by at least 30% or 40% but preferably 50%, 60% to 70% or even 100% orlonger, compared to the same treatment (e.g., chemotherapy) but withoutanti-HGF mAb. In addition or alternatively, treatment (e.g., standardchemotherapy) including the anti-HGF mAb may increase the completeresponse rate, partial response rate, or objective response rate(complete+partial) of patients with these tumors (e.g., ovarian, breast,lung, pancreas, brain and colon, especially when relapsed or refractory)by at least 30% or 40% but preferably 50%, 60% to 70% or even 100%compared to the same treatment (e.g., chemotherapy) but without theanti-HGF mAb. Optionally, treatment can inhibit tumor invasion, ormetastasis.

Typically, in a clinical trial (e.g., a phase II, phase II/III or phaseIII trial), the aforementioned increases in median progression-freesurvival and/or response rate of the patients treated with chemotherapyplus the anti-HGF mAb, relative to the control group of patientsreceiving chemotherapy alone (or plus placebo), will be statisticallysignificant, for example at the p=0.05 or 0.01 or even 0.001 level. Itwill also be understood by one of skill that the complete and partialresponse rates are determined by objective criteria commonly used inclinical trials for cancer, e.g., as listed or accepted by the NationalCancer Institute and/or Food and Drug Administration.

4. Other Methods

The anti-HGF mAbs of the invention also find use in diagnostic,prognostic and laboratory methods. They may be used to measure the levelof HGF in a tumor or in the circulation of a patient with a tumor, andtherefore to follow and guide treatment of the tumor. For example, atumor associated with high levels of HGF would be especially susceptibleto treatment with an anti-HGF mAb. In particular embodiments, the mAbscan be used in an ELISA or radioimmunoassay to measure the level of HGF,e.g., in a tumor biopsy specimen or in serum or in media supernatant ofHGF-secreting cells in cell culture. The use of two anti-HGF mAbsbinding to different epitopes (i.e., not competing for binding) will beespecially useful in developing a sensitive “sandwich” ELISA to detectHGF. For various assays, the mAb may be labeled with fluorescentmolecules, spin-labeled molecules, enzymes or radioisotypes, and may beprovided in the form of kit with all the necessary reagents to performthe assay for HGF. In other uses, the anti-HGF mAbs will be used topurify HGF, e.g., by affinity chromatography.

EXAMPLES

1. Generation of Anti-HGF mAbs

To generate mAbs which bind to and block the activities of human HGF,recombinant human HGF (rHGF) was first produced in a mammalianexpression system. cDNAs encoding the recombinant human HGF (rHGF) orrHGF-Flag peptide (8 amino acid residues of Flag attached to thec-terminus of HGF) were constructed in a pIND-inducible expressionvector (No et al., Proc. Natl. Acad. Sci. USA. 93:3346, 1996). ThesecDNAs were then transfected into EcR-293 human kidney fibroblast cells(Invitrogen) using Fugene transfection reagent (Roche). Stable celllines, H1-F11 and 24.1, secreting HGF and HGF-Flag respectively, wereselected in the presence of 600 μg/ml of G418 and 400 μg/ml of Zeocin(Invitrogen). H1-F11 and 24.1 were induced to secrete HGF and HGF-Flagby treatment with 4 μM of Ponasterone A (Invitrogen) for 4-5 days inserum free DMEM containing glutamine and antibiotics. After aggregateswere removed by centrifugation at 15,000 rpm for 30 min at 4° C., HGFsecreted into the culture supernatant was concentrated approximately100-fold using a membrane ultrafiltration cartridge with an MW 50,000cut-off filter [amicon Centriprep YM-50 filter followed by microconYM-50 filter (Millipore)]. Such concentrated H1F11 culture supernatantcontains ˜100 μg/ml of HGF and ˜120 μg/ml of bovine serum albumin.

Balb/c mice were immunized in each hind foot pad >10 times at one weekintervals, with 1-2 μg of purified rHGF (Pepro Tech) or 1-2 μg of rHGFplus 1-2 μg of BSA (concentrated H1-F11 culture supernatant) resuspendedin MPL-TDM (Ribi Immunochem. Research). Three days after the finalboost, popliteal lymph node cells were fused with murine myeloma cells,P3X63AgU.1 (ATCC CRL1597), using 35% polyethylene glycol. Hybridomaswere selected in HAT medium as described (Chuntharapai and Kim, J.Immunol. 163:766, 1997, which is incorporated herein by reference). Tendays after the fusion, hybridoma culture supernatants were screened in adirect HGF binding ELISA as well as in an HGF-Flag capture ELISA. Thelatter assay was used to further confirm the specificity of anti-HGFmAbs selected using the direct HGF binding ELISA and to select mAbs thatcan bind to HGF in solution phase. Blocking activities of selected mAbswere then determined in the HGF-Flag/ cMet-Fc binding ELISA and in theMDCK scatter assay as described (Jeffers et al., Proc. Natl. Acad. Sci.USA 95:14417, 1998). Selected hybridomas were cloned twice usinglimiting dilution techniques. The isotype of mAbs were determined usingan isotyping kit (Zymed). Ascites of selected mAbs were raised andpurified using ImmunoPure (A/G) IgG Purification Kit (Pierce). Alsobiotinylated mAbs were prepared using EZ-sulfo-NHS-LC-Biotin accordingto the instructions provided by Pierce. Each of the assays referred tohere is described in more detail below. For the direct HGF bindingELISA, microtiter plates (Maxisorb; Nunc) are coated with 50 μl/well ofH1-F11 culture supernatants containing rHGF, diluted in PBS at a 1:2ratio of HGF/PBS, overnight at 4° C. After washing the plate, thenonspecific binding sites are blocked with PBS containing 2% skim milkfor 1 hr at room temperature (RT). After washing the plate, 50 μl/wellof purified mAbs or hybridoma culture supernatants are added to eachwell for 1 hr. After washing, plates are then incubated with 50 μl/wellof 1 μg/ml of HRP-goat anti-mouse IgG (HRP-GαMIgG, Cappel) for 1 hr. Thebound HRP-GαMIgG is detected by the addition of the tetramethylbenzidinesubstrate (Sigma). The reaction is stopped by the addition of 1N H₂SO4and the plates are then read at 450 nm using an ELISA plate reader.Washes are carried out 3 times in wash buffer (PBS containing 0.05%Tween 20).

For the HGF-Flag capture ELISA, microtiter plates are coated with 50μl/well of 2 μg/ml of goat antibodies specific to the Fc portion ofmouse IgG (GαMIgG-Fc) in PBS overnight at 4° C. and blocked with 2% skimmilk for 1 hr at RT. After washing, the plates are incubated with 50μl/well of purified mAbs or hybridoma culture supernatants for 1 hr.After washing, plates are then incubated with 50 μl/well of 24.1 cellculture supernatant containing rHGF-Flag. After washing, plates are thenincubated with 50 μl/well of HRP-M2 anti-Flag mAb (Invitrogen) in thepresence of 15 μg/ml of murine IgG. The bound HRP-anti-Flag M2 isdetected by the addition of the substrate as described above. Washes arecarried out 3 times in wash buffer.

At least three mAbs, designated L1H4, L2C7 and L2G7, obtained fromhybridomas generated by immunizing the Balb/c mice with rHGF inconcentrated H1-F11 culture supernatant as described above, showedbinding in both the direct rHGF binding ELISA and the HGF-Flag captureELISA and were selected for further study. These hybridomas were thencloned twice, ascites were raised in mice by standard methods, and mAbswere purified using a protein G/A column. Their isotypes were determinedusing an isotyping kit (Zymed Lab). The L2G7 hybridoma has beendeposited on Apr. 29, 2003 with the American Type Culture Collection,P.O. Box 1549 Manassas, Va. 20108, as ATCC Number PTA-5162 under theBudapest Treaty. These deposit will be maintained at an authorizeddepository and replaced in the event of mutation, nonviability ordestruction for a period of at least five years after the most recentrequest for release of a sample was received by the depository, for aperiod of at least thirty years after the date of the deposit, or duringthe enforceable life of the related patent, whichever period is longest.All restrictions on the availability to the public of these cell lineswill be irrevocably removed upon the issuance of a patent from theapplication.

Once a single, archtypal anti-human-HGF mAb, for example L2G7, has beenisolated that has the desired properties described herein ofneutralizing HGF in vitro and/or inhibiting (e.g., completely) tumorgrowth in vivo, it is straightforward to generate other mAbs withsimilar properties, by using art-known methods. For example, mice may beimmunized with HGF as described above, hybridomas produced, and theresulting mAbs screened for the ability to compete with the archtypalmAb for binding to HGF. Alternatively, the method of Jespers et al.,Biotechnology 12:899, 1994, which is incorporated herein by reference,may be used to guide the selection of mAbs having the same epitope andtherefore similar properties to the archtypal mAb, e.g., L2G7. Usingphage display, first the heavy chain of the archtypal antibody is pairedwith a repertoire of (preferably human) light chains to select anHGF-binding mAb, and then the new light chain is paired with arepertoire of (preferably human) heavy chains to select a (preferablyhuman) HGF-binding mAb having the same epitope as the archtypal mAb.

2. Characterization of Anti-HGF mAbs In Vitro

The binding epitopes of the antibodies were partially characterized by acompetitive binding ELISA in which a 100× excess of unlabeled mAb wasused to compete with the binding of the same or another biotinylated mAbin the HGF binding ELISA. FIG. 4 shows that the binding of the anti-HGFmAbs, L1H4 and L2G7, was inhibited only by themselves, suggesting thatthey recognize unique epitopes. The binding of L2C7 was inhibited byL2G7 but not by L1H4. This suggests that the L2C7 epitope overlaps withthat of L2G7 but not of L1H4. However, L2C7 was not able to inhibit thebinding of L2G7, suggesting that the L2C7 and L2G7 epitopes overlap butare distinct, and/or the affinity of L2C7 is much lower than that ofL2G7. The epitopes of L1H4, L2C7 and L2G7 are respectively designated A,B and C.

The relative binding abilities of the three anti-HGF mAbs were measuredusing purified antibodies in the direct HGF binding ELISA, in which rHGFis first bound to the plate. In this assay, L2C7 and L2G7 bound betterthan L1H4 (FIG. 5). The ability of the mAbs to bind rHGF-Flag insolution was also determined, using the HGF-Flag capture ELISA. Allthree mAbs were able to capture rHGF-Flag in solution phase but mAb L2G7was more effective than the others (FIG. 6). These results suggest thatmAb L2G7 has the highest binding affinity to HGF among the three mAbs.

One of the biological activities of HGF is the ability to bind to itsreceptor cMet, so the ability of the anti-HGF mAbs to inhibit binding ofHGF to cMET was assayed. For this assay, cMet-Fc was first produced bytransfecting human fibroblast 293 cells with cDNA encoding residues1-929 ECD of cMet linked with the Fc portion of human IgG1 (residues 216to 446) as described by Mark et al., J. Biol. Chem. 267:26166, 1992 inthe pDisplay expression vector (Invitrogen). Microtiter plates arecoated with 50 μl/well of 2 μg/ml of goat antibodies specific to the Fcportion of human IgG (GαHIgG-Fc) in PBS overnight at 4° C. and blockedwith 2% BSA for 1 hr at RT. After washing the plates, 50 μl of culturesupernatant of 293 transfected with cMet-Fc cDNA is added to each wellfor 1 hr at RT. After washing the plates, 50 μl/well of 24.1 cellculture supernatant containing rHGF-Flag, preincubated with variousconcentrations of mAbs, is added to each well for 1 hr. After washing,plates are then incubated with 50 μl/well of HRP-M2 anti-Flag mAb(Invitrogen). The bound HRP-anti-Flag M2 is detected by the addition ofthe substrate as described above. Washes are carried out 3 times in washbuffer.

In this HGF-Flag/cMet-Fc binding inhibition assay, all three mAbsdemonstrated some degrees of inhibition while an Ig control antibody didnot (FIG. 7). MAb L2G7 at ≧1 μg/ml and mAb L1H4 at 50 μg/ml completelyabolished the binding of rHGF-Flag to cMet-Fc; mAb L2C7 even at 50 μg/mlgave only 85% inhibition. Hence, mAb L2G7 was much more potent ininhibiting the interaction of rHGF-Flag with cMet-Fc (and thereforepresumably HGF with its receptor cMet) than the other antibodies,consistent with its putatively greater affinity for HGF.

Since the receptor protein used in cMet-Fc/HGF-Flag binding ELISA is asoluble receptor protein, its conformation may be different from that ofthe natural membrane bound receptor. Furthermore, HGF binds to HSPG inaddition to cMet and it is known that the HSPG-HGF interaction enhancesvarious HGF activities. Thus, mAbs blocking the interaction of HGF withsoluble cMet may not necessarily have the capacity to neutralize HGFbioactivities on the cells. Thus, it is important to further confirm theblocking activities of mAbs in selected biological systems. HGF is knownto be a potent scattering factor. Thus, the neutralizing activity of theanti-HGF mAbs was also determined using the Madin-Darby canine kidney(MDCK cells obtained from ATCC) scatter assay as described (Jeffers etal., Proc. Natl. Acad. Sci. USA 95:14417, 1998). MDCK cells grown inDMEM supplemented with 5% FCS are plated at 10³ cells/100 μl/well in thepresence of predetermined concentrations of rHGF with or without mAbs inDMEM with 5% FCS. After 2 days incubation at 37° C. in 5% CO₂, cells arethen washed in PBS, fixed in 2% formaldehyde for 10 min at RT. Afterwashing in PBS cells are stained with 0.5% crystal violet in 50% ethanol(v/v) for 10 min at RT. Scattering activity is determined by microscopicexamination.

Culture supernatant of the H1-F11 clone secreting HGF, described above,was used as the source of HGF in the scatter assay. As little as 1:80dilution of H1-F11 culture supernatant induced the scattering and growthof MDCK cells. However, the scattering assays were carried out using a1:20 dilution of H1-F11 culture supernatant (˜3 μg/ml). MAb L2G7 even ata 1:5 molar ratio of HGF/mAb inhibited the HGF induced scattering ofMDCK by itself (FIG. 8), conclusively demonstrating that mAb L2G7 isindeed a neutralizing mAb. mAb L1H4 at ≧20 μg/ml could also neutralizescattering of MDCK induced by HGF, while mAb L2C7 even at 20 μg/ml gaveonly a partial neutralizing activity (data not shown).

The various characteristics of the three anti-HGF antibodies determinedin the assays above are summarized in the Table 1.

TABLE 1 Characterization of mAbs to HGF Binding Block Block mAb IsotypeEpitope HGF/cMet-Fc binding MDCK scattering L1H4 G1, κ A Weak Block +L2C7 G2b, κ B Partial Block +/− L2G7 G2a, κ C Strong Block +++

HGF is a member of the heparin binding growth factor family includingfibroblast growth factor (FGF) and vascular endothelial growth factor(VEGF). Also, HGF has ˜40% overall sequence similarity with plasminogen(Nakamura et al., Nature. 342:440, 1989) and shares a similar domainstructure with macrophage stimulating protein (MSF, Wang et al., Scand.J. Immunol. 56:545, 2002). Thus, the binding specificity of the anti-HGFantibodies must be determined. The binding of anti-HGF mAbs to these HGFrelated proteins (available from R&D systems) is assayed using a directbinding ELISA similar to the one for HGF described above. MAb L2G7, mAbL2C7 and mAb L1H4 will not significantly bind to these proteins,demonstrating their specificity for HGF.

3. Ability of Anti-HGF MAbs to Inhibit Tumor-Promoting BiologicalActivities of HGF

HGF has a number of biological activities that make it likely that itplays a role in the growth and invasiveness of certain human tumors. Onesuch activity of HGF is as a powerful mitogen for hepatocytes and otherepithelial cells (Rubin et al., Proc. Natl. Acad. Sci. USA. 88:415,1991). Thus, to further prove the neutralizing activity of the anti-HGFmAbs, the effects of the mAbs on the HGF-induced proliferation of 4MBr-5monkey epithelial cells (ATCC) or rat hepatocytes are determined.Hepatocytes are isolated according to a method described by Garrison andHaynes, J. Biol. Chem. 269:4264, 1985. Cells are resuspended at 5×10⁴cells/ml in DMEM containing 5% FCS and stimulated with a predeterminedconcentration of HGF with various concentration of mAbs. After 2½ daysincubation at 37° C. in 5% CO₂, the level of cell proliferation isdetermined by the addition of ³H-thymidine for 4 hrs. Cells areharvested using an automated cell harvester and the level of³H-thymidine incorporated is determined on a scintillation counter. Atsufficient concentrations, mAb L2G7 may largely or completely inhibitHGF-induced proliferation of the cells, and mAbs L2C7 and L1H4 may atleast partially inhibit proliferation. These antibodies may also inhibitthe HGF-induced proliferation of other epithelial cell lines.

For example, the inhibitory activity of L2G7 on the HGF-inducedproliferation of mink lung Mv 1 Lu cells was determined (Borset et al.,J. Immunol. Methods 189:59, 1996). Cells grown in DMEM containing 10%FCS are harvested by treatment with EDTA/trysin. After washing, thecells are resuspended at 5×10⁴ cells/ml in serum free DMEM with apredetermined concentration (50 ng/ml) of HGF +/− various concentrationsof mAb. After 1 day incubation at 37° C. in 5% CO₂, the level of cellproliferation is determined by the addition of 1 μCi of ³H-thymidine foran additional 24 hr. Cells are harvested onto glass-fiber filters usingan automated cell harvester and the level of ³H-thymidine incorporatedis determined on a scintillation counter. FIG. 9 shows that the additionof 100-fold higher molar concentration of L2G7 mAb completely inhibitedthe proliferative response of Mv 1 Lu cells. Indeed, L2G7 even at a3-fold molar ratio of mAb to HGF showed complete inhibition, whilecontrol IgG showed no inhibition even at 100-fold molar excess.

HGF is also reported to be a potent angiogenesis factor (Bussolino etal., J. Cell Biol. 119:629, 1992; Cherrington et al., Adv. Cancer Res.79:1, 2000), and angiogenesis, the formation of new blood vessels, isbelieved to be essential to the growth of tumors. Therefore, the abilityof the anti-HGF mAbs to inhibit the angiogenic properties of HGF isshown in three assays: (i) proliferation of human vascular endothelialcells (HUVEC), (ii) tube formation of HUVEC, and (iii) development ofnew blood vessels on the chick embryo chorioallantoic membrane (CAM).Since HGF has been shown to synergize with VEGF in angiogenesis (Xin etal., Am. J. Pathol. 158:1111, 2001), these assays may be performed bothin the presence and absence of VEGF.

The HUVEC proliferation assay is performed as described with amodification (Conn et al., Proc. Natl. Acad. Sci. USA 87:1323, 1990).HUVEC cells obtained from Clonetics are grown in Endothelial GrowthMedium (EBM-2) containing 10% FCS plus endothelial cell growthsupplements provided by Clonetics. Preferably cells from passages 4 to 7are used in this study. The cells are resuspended to be 10⁵ cells/ml inmedium-199 containing antibiotics, 10 mM HEPES and 10% FCS (assaymedium). HUVEC cells (50 ill/well) are added to microtiter wellscontaining a suitable concentration of HGF with various concentrationsof anti-HGF mAbs for 1 hr at 37° C. After cells are incubated for 72 hrat 37° C. in 5% CO₂, the level of cell proliferation is determined byincorporation of ³H-thymidine for 4 hrs. At sufficient concentrations,mAb L2G7 will largely or completely inhibit HGF-induced proliferation ofthe HUVEC, and mAbs L2C7 and L1H4 may at least partially inhibitproliferation.

Alternatively, the level of cell proliferation may be determined by thewell-known calorimetric MTT assay. The HUVECs (10⁴ cells/100 μl/well)are grown in serum free medium for 24 hr, and then incubated with 100 μlof of 50 ng/ml of HGF (predetermined to be a suboptimal amount) withvarious concentrations of mAb L2G7 for 72 hr. MTT solution (5 mg/ml) isadded to each well (20 μl/200 μl medium) for 4 hr. Then 100 μlmedium/well is removed and mixed with 100 μl/well of acidified isopropylalcohol (0.04N HCl in isopropyl). The plates are read on an ELISA readerat 560 nm. The % maximum response is calculated as follows: [OD ofHGF+mAB treated cells−OD of untreated cells]/[OD of HGF treated cells−ODof untreated cells] ×100. FIG. 10 shows that even a 2-fold molar excessof L2G7 mAB largely blocks the proliferation of HUVEC in response toHGF.

The endothelial tube assay is carried out essentially as described(Matsumura, et al., J. Immunol. 158:3408, 2001; Xin et al., Am. J.Pathol. 158:1111, 2001). HUVEC (Clonetics) from passage 4-7 are grown inClonetics EGM medium supplemented with 10% FBS and endothelial cellgrowth supplements. Plates are coated with Matrigel (BD Biosciences)according to the manufacture's instructions at 37° C. for 30 min, andthe cells are seeded as 3×10⁶ cells/ml in 1× basal medium with HGF andvarious concentrations of anti-HGF mAbs. Tube formation is evaluatedunder microscope at low-power (10×) magnification. At sufficientconcentrations, mAb L2G7 will largely or completely inhibit HGF-inducedendothelial tube formation, and mAbs L2C7 and L1H4 may at leastpartially inhibit it.

The chick embryo chorioallantoic membrane (CAM) assay is performedessentially as described (Kim et al, Nature 362:841, 1992). Three-dayold chicken embryos are removed from their shells and grown in petridishes in 5% CO₂ at 37° C. Seven days later, dried methylcellulose discscontaining HGF with various concentrations of anti-HGF mAbs are layeredonto the CAM. The methylcellulose discs are prepared by mixing 5 μl of1.5% methylcellulose in PBS with 5 μl of HGF preincubated with mAbs.Three days later the development of blood vessels around methylcellulosediscs are examined. At sufficient concentrations, mAb L2G7 will largelyor completely inhibit such blood vessel formation, and mAbs L2C7 andL1H4 may at least partially inhibit it.

HGF is also reported to promote tumor growth (Comoglio and Trusolino, J.Clin. Invest. 109:857, 2002). The ability of the anti-HGF antibodies toinhibit this activity is shown in two steps. First, a number of tumorcell lines are examined for their ability to secrete HGF and proliferatein response to HGF since HGF may be an autocrine growth factor for someof these cells. These cell lines include a panel of human tumor celllines known to express HGF and cMet (Koochekpour et al., Cancer Res.57:5391, 1997; Wang et al., J. Cell Biol. 153:1023, 2001). Specific celllines to be tested include U-118 glioma, HCT116 colon carcinoma, A549lung carcinoma and A431 epidermoid carcinoma cells, all available fromthe ATCC. Once such tumor cell lines are identified, the effect ofanti-HGF mAbs on the proliferative response to HGF of these cells isdetermined, using methods similar to those described above. Atsufficient concentrations, mAb L2G7 will largely or completely inhibitHGF-induced proliferation of many or all of these cell lines, and mAbsL2C7 and L1H4 may at least partially inhibit proliferation.

For example, human HCT116 tumor cells are seeded into 96-well microtiterplates at 5×10³ cells/well in 200 μl of DMEM plus 5% FCS. After 24 hrincubation at 37° C. in 5% CO₂, cells are washed with PBS and incubatedin serum free DMEM for 48 hr. Cells are then incubated with 100 ng/ml ofHGF +/−20 μg/ml of mAbs in DMEM for another 20 hr. As controls, cellsgrown in DMEM alone or DMEM plus 10% FCS are included. At the end of theincubation, levels of cell proliferation are determined by incorporationof ³H-thymidine for 4 hr. The result of such an experiment was carriedout in triplicates is shown in FIG. 11. HGF induced a moderateproliferation of the HCT116 cells, which was completely abolished byaddition of L2G7 antibody (but not by the less potent L1H4 antibody).

In all the assays described above, each anti-HGF antibody willneutralize or inhibit activity when used alone without other antagonistsof HGF, i.e., as a single agent, but additive or synergistic effects maybe achieved by administering the antibody in conjunction with otheranti-HGF antibodies or other active agents.

4. Ability of anti-HGF mAbs to Inhibit Tumor Growth In Vivo

The ability of the anti-HGF antibodies to inhibit human tumor growth isdemonstrated in xenograft models in immunodeficient mice or otherrodents such as rat. Ilustrative but not limiting examples ofimmunodeficient strains of mice that can be used are nude mice such asCD-1 nude, Nu/Nu, Balb/c nude, NIH-III (NIH-bg-nu-xid BR); scid micesuch as Fox Chase SCID (C.B-17 SCID), Fox Chase outbred SCID and SCIDBeige; mice deficient in RAG enzyme; as well as nude rats. Experimentsare carried out as described previously (Kim et al., Nature 362:841,1992, which is incorporated herein by reference). Human tumor cellsgrown in complete DMEM medium are harvested in HBSS. Femaleimmunodeficient, e.g., athymic nude mice (4-6 wks old) are injected s.c.with typically 5×10⁶ cells in 0.2 ml of HBSS in the dorsal areas. Whenthe tumor size reaches 50-100 mm³, the mice are grouped randomly andappropriate amounts of the anti-HGF and control mAbs (typically between0.1 and 1.0 mg, e.g. 0.5 mg) are administered i.p. once, twice or threetimes per week in a volume of, e.g., 0.1 ml, for e.g., 1, 2, 3, or 4weeks or the duration of the experiment. Tumor sizes are determinedtypically twice a week by measuring in two dimensions [length (a) andwidth (b)]. Tumor volume is calculated according to V=ab²/2 andexpressed as mean tumor volume±SEM. The number of mice in each treatmentgroup is at least 3, but more often between 5 and 10, e.g., 7.Statistical analysis may be performed using, e.g., Student's t test. Ina variation of this experiment, administration of the antibody beginssimultaneously or shortly after injection of the tumor cells. The effectof the antibody may also be measured by prolongation of the survival ofthe mice, or increase in percent of the mice surviving.

Various tumor cell lines known to secrete or respond to HGF are used inseparate experiments, for example U118 human glioblastoma cells, and/orHCT116 human colon tumor cells. Preferred antibodies of the invention,such as human-like and reduced-immunogenicity antibodies and the L2G7antibody and its chimeric and humanized forms and antibodies with thesame epitope as L2G7, when used as a single agent, will inhibit growthof tumors by at least 25%, but possibly 40% or 50%, and as much as 75%or 90% or greater, or even completely inhibit tumor growth after someperiod of time or cause tumor regression or disappearance. Thisinhibition will take place for at least tumor cell line such as U118 inat least one mouse strain such as NIH III Beige/Nude, but preferablywill occur for 2, 3, several, many, or even essentially allHGF-expressing tumor cell lines of a particular (e.g., glioma) or anytype, when tested in one or more immunodeficient mouse strains that donot generate a neutralizing antibody response against the injectedantibody. Treatment with some preferred antibodies in one or more of thexenograft models leads to the indefinite survival of 50%, 75%, 90% oreven essentially all mice, who would otherwise die or need to besacrificed because of growth of their tumor.

For example, such an experiment was performed with U-118 glioblastomacells, grown in DMEM medium with FCS and harvested in HBSS. Female NIHIII Beige/Nude mice (4-6 wks old) are injected s.c. with 10⁶ cells in0.2 ml of HBSS in the dorsal areas. When the tumor size reaches ˜50 mm ,the mice are grouped randomly into 2 groups of 6 mice each, and 200 μgof the L2G7 mAb (treatment group) or of PBS (control group) are giveni.p. twice a week in a volume of 0.1 ml. Tumor sizes are determinedtwice a week as described above. At the end of the experiment, thetumors are excised and weighed. FIG. 12 shows that treatment with L2G7completely inhibited tumor growth.

Similar tumor inhibition experiments are performed with the anti-HGFantibody administered in combination one or more chemotherapeutic agentssuch as 5-FU (5-fluorouracil) or CPT-11 (Camptosar) to which the tumortype is expected to be responsive, as described by Ashkenize et al., J.Clin. Invest. 104:155, 1999. The combination of the antibody andchemotherapeutic drug may produce a greater inhibition of \tumor growththan either agent alone. The effect may be additive or synergistic, andstrongly inhibit growth, e.g. by 80% or 90% or more, or even cause tumorregression or disappearance. The anti-HGF antibody may also beadministered in combination with an antibody against another growth orangiogenic factor, for example anti-VEGF, and additive or synergisticgrowth inhibition and/or tumor regression or disappearance is expected.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the invention. Unlessotherwise apparent from the context any step, element, embodiment,feature or aspect of the invention can be used with any other.

All publications, patents and patent applications cited are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent and patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety for all purposes.

1. A monoclonal antibody (mAb) that binds and neutralizes humanHepatocyte Growth Factor (HGF).
 2. The mAb of claim 1 which is chimeric.3. The mAb of claim 1 which is humanized.
 4. The mAb of claim 1 which ishuman.
 5. The mAb of claim 1 which inhibits binding of HGF to cMet by atleast 50%.
 6. The mAb of claim 1 which inhibits HGF-induced scatteringof Madin-Darby canine kidney cells.
 7. (canceled)
 8. The mAb of claim 1which inhibits HGF-induced angiogenesis.
 9. The mAb of claim 1 whichneutralizes all biological activities of HGF.
 10. The mAb of claim 1which inhibits growth of a human tumor xenograft in a mouse.
 11. The mAbof claim 10 which completely inhibits growth of a human tumor xenograftin a mouse.
 12. The mAb of claim 1 which is a Fab or F(ab′)₂ fragment orsingle-chain antibody.
 13. The mAb of claim 1 which specifically bindsHGF with a binding affinity of at least 10⁸ M⁻¹.
 14. A chimeric orhumanized L2G7 mAb.
 15. An antibody that competes for binding to humanHGF with an antibody of claim
 14. 16. A cell line producing a mAb ofclaim
 1. 17. A pharmaceutical composition comprising a mAb of claim 14.18. A method of treating cancer in a patient comprising administering tothe patient a pharmaceutical composition comprising a neutralizinganti-HGF mAb.
 19. A method of claim 18 wherein said cancer isglioblastoma.
 20. The method of claim 18 wherein said mAb is a chimericor humanized L2G7 mAb.